I'm first time here and I'm really frustrated by cloning now.
I've been trying to clone a 4kb insert into a vector for almost 2 months.
What I did is....
1) I cut both plasmid and insert with KpnI-HF and XmaI.
2) Purify the digested product with Quiagen purification kit.
3) Ligate them using Invitrogen T4 ligase using insert:vector ratios like 3:1 or 5:1.
4) Transform the ligated product to DH5-alpha competent cells.
I always do a negative control for transformation and each time I get no colonies on negative control plates but with lots of colonies on the plate transformed with the supposedly ligated vector.
The weird thing is if I take 16 colonies. All of them only have vectors without insert.
The question is I don't know how this is possible because the cut vectors should not reclose to themselves and produce vectors without inserts.
I've tests to make sure both enzymes work: I tried to cut the vector with KpnI-HF only, with XmaI only, with both and with none of them All of them produce a clear band at the right size. the last one has a supercoil and the others are linearized forms.
In addition, I've made sure that the enzymes should be able to digest all of the vectors I added in so there should not be undigested vectors in the product I used for transformation.
Anyone got any clue about what's happening here? Please give me some suggestion!! Thanks a million!!!
Edited by AllisonWu, 07 October 2010 - 06:11 PM.